Sec Mutants/Proteins (Molecular Biology)

A compelling approach to any biological process is to generate mutants that affect it. One such process is protein secretion. Almost all cells in almost all organisms are capable of secreting proteins. Thus it is possible to study secretion in two of the organisms most amenable to genetic analysis, Escherichia coli and Saccharomyces cerevisiae. Mutants in bacteria and yeast with defective secretion have been called sec mutants.

Proteins are secreted from bacteria by translocating them in an unfolded state directly from the cytoplasm across the plasma membrane into extracellular space. The sec mutations in E. coli, therefore, affect the unfolding process (secB, Table 1), the translocase or pore in the plasma membrane through which the nascent protein passes (secY, secE, secG, etc.), and the motor that drives the protein through the pore (sec A).

Yeast, like all eukaryotic cells, translocate newly synthesized proteins across the membrane of the endoplasmic reticulum (ER). This process closely resembles translocation across the plasma membrane in bacteria. The protein must be kept unfolded by a molecular chaperone (Table 1) and is translocated through a pore whose basic constituents are homologous to the secY, secE, and secG proteins in E. coli (Table 2). There is no eukaryotic equivalent of the ATP-driven (adenosine triphosphates) translocating machinery, encoded in E. coli by secA.

Table 1. Mutations Affecting Chaperones

Name	Organism	Location	Notes
SecB	E. coli	cytoplasm	Delivers protein to secB
DNAJK	E. coli	cytoplasm	?
GroE	E. coli	cytoplasm	Affects pre-b-lactamase. GroEL and GroES together form a complex multisubunit structure (chaperonin)
Ffh	E. coli	cytoplasm	Part of bacterial ribonucleoprotein signal recognition particle (SRP) complex; interacts with 4-5S RNA
sec65	yeast	cytoplasm	Homologue of 19-kDa protein of mammalian SRP.Required for assembly of integral membrane proteins
SRP54	Yeast	cytoplasm	Homologue of 54-kDa protein of mammalian SRP,also a GTPase
Ssa 1-4	Yeast	cytoplasm	Members of yeast heat-shock protein family required for ATP-dependent posttranslational translocation
Kar2p	Yeast	Lumen of ER	Analogous to mammalian BiP, required for translocation into ER
PDIl	Yeast	Lumen of ER	A protein disulfide isomerase that catalyzes disulfide bond formation asthe nascent chain enters the ER
FkB2	Yeast	Lumen of ER	Peptidyl proline cis-trans isomerase

Table 2. Mutations Affecting Membrane Translocation

Name

Organism

Location

Notes

secY,	E. coli	Plasma	Forms transmembrane channel
E, G		membrane
secA	E. coli	cytoplasm	ATP-driven shuttle, pushing protein
			through translocon.Recognizes newly
			synthesized protein, complexed with secB
secD,	E. coli	Plasma	Accessory proteins for bacterial translocon
secF		membrane
sec61,	Yeast	ER	Forms transmembrane channel
62, 63
			Homologue of mammalian 61p, a, b, g
SRP	Yeast	ER	A GTPase that acts as receptor for a signal
101		membrane	recognitionparticle (SRP); homologue of
			mammalian a-subunits
ft-sY	E. coli	?	Bacterial homologue of SRP receptor

In eukaryotes, protein secretion also requires membrane traffic from the endoplasmic reticulum to the Golgi complex, and from there to the plasma membrane. Each of the two membrane trafficking steps involves segregating cargo proteins from endogenous proteins, forming a carrier vesicle, and fusing the carrier vesicle with the appropriate target. The same categories of proteins are involved in each step: namely, sortases, coating molecules, and SNARE complexes to allow fusion. There are also conserved Gtpases (guanosine triphosphate hydrolases) that regulate the coating and targeting steps. Finally, the movement of carrier vesicles sometimes requires cytoskeleton elements, and so secretion can be perturbed by mutations that affect the cytoskeleton.

The steps at which sec mutants act are described in the entries protein secretion, exocytosis, and secretory vesicles/granules. Indeed, much of our understanding of these topics has come from analysis of mutants defective in secretion. For convenience, mutations are listed here by their function. It is doubtful that all mutations that affect secretion have been identified, but the overall patterns seem to be clear.

In several cases, yeast mutations that affect secretion were not isolated through a direct screen for a secretion deficiency. Instead, they were selected as mutations that acted as suppressors of a deficiency, or as giving synthetic lethality when combined with a weak secretion-deficiency allele. In other cases, they were identified by homology to a mammalian protein of known function. Finally, some were products of other selections but were found also to have a "sec" phenotype. Thus not all of the mutants listed in Tables 1 and 2 have the conventional designation of sec followed by a number or letter.

1. Chaperones

Molecular chaperones are proteins that help keep newly synthesized proteins unfolded until they pass through the transmembrane channel and then help fold them correctly after translocation. Representative examples are given in Table 1.

2. Membrane Translocation

The newly synthesized proteins cross membranes via a proteinaceous channel. Proteins that recognize the signal peptides help present the newly synthesized protein to the channel. Mutations are found in channels and in the receptors that recognize signal peptides. 3. Budding from the endoplasmic reticulum

In eukaryotes, newly synthesized proteins leave the ER in a coated vesicle. The COPII coatomers are clearly involved in the ER to Golgi pathway. Since the ret1 mutation blocks retrieval of dilysine-containing ER proteins from the Golgi, COPIs are involved in intra-Golgi or Golgi to ER traffic. Mutants in COPI may only affect ER to Golgi traffic indirectly. Cargo can be selected from ER-resident proteins by specific "sortases" such as SHR3 or emp24. (See Table 3.)

Table 3. Mutations in ER to Golgi Traffic

Name	Organism	Location	Notes
SHR3	Yeast	ER membrane	A sortase required specifically for amino-acid permease export from the ER
emp24	yeast	ER membrane	Required for invertase secretion. Believed to be a carrier molecule
sec23/24	Yeast	Cytosol	Form a heterodimer; sec23p has GAP activity. With sec 13/31, form a COPII coat on ER-budding vesicles
sec 13/31	Yeast	Cytosol	Form heterodimers. With sec23/24p, form a COPII coat on ER-budding vesicles
saur1	yeast	Cytosol	A yeast GTPase, homologous to ARF1, which mediates coating and uncoating
sec 12	yeast	ER membrane	Sar1 exchange factor
RET 1/sec26	yeast	ER membrane	Homolog of the a-, b-, b’-, and g-subunits of sec27, sec21 mammalian coatomer (COPI)
ARF1/2	yeast	ER membrane	Yeast homologues of the small mammalian GTPase, ARF1

4. Fusion of ER-Derived Carrier Vesicles with Golgi Membranes

ER-derived vesicles fuse with the cis-Golgi network. Recognition appears to be mediated by specific SNAREs. There is also a parallel recognition pathway involving another small GTPase, ypt1 or rab1, which, like all rab proteins, has an isoprene addition to its C-terminal tail. (See Table 4.)

Table 4. Mutations Affecting Fusion with Golgi Membranes

Name

Organism

Location

Notes

Bctl	Yeast	ER carrier vesicle	Similar in structure to a v-SNARE
Bosl	Yeast	ER carrier vesicle	Suppressor of betl mutations. Required for fusion competence. Homologue of synaptobrevin/VAMP
sec22	Yeast	ER carrier vesicle	v-SNARE
yptl	Yeast	ER carrier vesicle	A small ras-like GTPase, homologue of mammalianrabl
bet2	Yeast	cytoplasm	A geranyl geranyl transferase for yptl
sec 18	Yeast	cytoplasm	An ATPase required for all fusion reactions; homologue of NSF (n-ethylmaleimide sensitive factor)
sec 17	Yeast	cytoplasm	Peripheral membrane protein that binds secl8p to membranes; homologue of a-SNAP
end2	Yeast	Golgi membranes	Receptor for proteins carrying HDEL sequence; homologue of mammalian KDEL receptor
sed5	Yeast	Post-ER structures	A putative t-SNARE; homologue of mammalian syntaxin
slyl	Yeast	Golgi membranes	Homologue of secl,which interacts with syntaxin at the cell surface

5. Golgi to Plasma Membrane Traffic

The basic requirements for transfer between Golgi and plasma membrane are identical to those that are involved in ER to Golgi traffic, since the steps of vesicle formation and selective vesicle fusion are common to both. Sometimes the same proteins—for example, sec17p (a-SNAP), sec18p (NSF) (NEM-sensitive factor), and ARF1 (ADP-ribosylation factor)—are common to both steps. Table 5 shows mutations that affect exocytoses.

Table 5. Mutations that affect Exocytosis

Name	Organism	Location	Notes
Snc l,2	Yeast	Secretory vesicles	Homologues of VAMP/synaptobrevin. Redundant geneproducts
sec7	Yeast	Golgi	Peripheral membrane protein believed to coat Golgi-derived carrier vesicles
kex2	Yeast	Golgi membrane	Serine proteinase that cleaves protein precursors in lumen of Golgi that have Lys-Arg or Arg-Arg sequences. Homologue of mammalian furin
kexl	Yeast	Golgi	Removes the two basic residues after

		membranes	kex2 has made its proteolytic cleavage
sec1	Yeast	Plasma membrane	Homologue of nsec 1, which binds tightly to syntaxin. Mutants cause secretory vesicle accumulation
sec9	Yeast	Plasma membrane	Homologue of SNAP-25
sec8,	Yeast	Cytoplasm	Forms a large 19S complex required for sec 15, etc. exocytosis, in addition to SNAREs
sec4	Yeast	Secretory vesicles	A small GTPase of the ras family
DSS4	Yeast		A guanine nucleotide exchangeprotein acting on sec4
act1	Yeast	Cytoplasmic cytoskeleton	Mutants cause accumulation of 100-nm secretoryvesicles
MYO-2Yeast		Cytoplasmic cytoskeleton	A novel yeast myosin of the class V-type; causes accumulation of 100-nm vesicles