Table 1.

Summary of Vps26 mutant phenotypes

Mutant Number	Mutations(human/yeast equivalents)	Vps35 binding in two- hybrid assay a	Assembly with Vps35, Vps29 in vivo b	Endosomal localization in cells	Rescue of CPY processing in VPS26Δ yeast d	Rescue of CPY secretion in VPS26Δ yeast e
–	WT	++	+	+	+	+
1	L431A V144D K145D	++
2	E13A D15A	++
3	K133D T135D V137D	++
4	R69A E71A	++	+	+
5	L97D	++
6	R127E R129E F131E	++
7	F79D	++
8	R271E F273E	++
9	K214E I216E	++
10	T225D T227D	++
11	I230D Y233D	++
12	L250D F251D	++
13	F197D L198D	++
14	V200D I204D	++
15	I246D	++
16	I235D M236D/I318D M319D	–	–	–	–	–
17	R284E R285E F287E	++
18	Δ238–246 GG/Δ321–328 GG	–	–	–	–	–
19	V190D V192D K194D	++
20	E177A E179A K182D	++
21	E165D K167A	++
22	E171A D172A	++
23	E281A E182A D183A R284A	++
24	S49A K51D N53A	++
25	V137D R139D L140D	++
26	T102D Q103D R105D	++
27	D108A E110A M112D	++
28	V53A A55D K57D	++
29	K61A R62D E64D	++
30	E234A D237A	++
31	I235D	+
32	M236D	+
33	G238P/G321P	±	–	–	–	–
34	238–246 polyS/321–328 polyS	±	–	–	–	–
35	Y121F/Y150F			+		+
36	N125R/N155D			+		+
37	E215R/E298R			+		+
38	R296E/R377E			+		+

^aYeast two-hybrid assays were performed as in Fig. 5; ++, normal growth; +, reduced growth in the presence of 10mM 3-AT; ±, lack of growth in the presence of 10mM 3-AT, -, no growth.

^bCo-precipitation of Vps35 and Vps29 with wild-type or mutant Vps26-myc expressed by transfection in HeLa cells was performed as in Fig. 6; +, normal assembly; -, reduced or no assembly.

^cCo-localization (+) or lack thereof (−) of wild-type or mutant Vps26-myc with SNX1 on endosomes in HeLa cells analyzed as in Fig. 6. For each condition, 50–75 cells from three independent experiments were examined.

^d,eCPY processing and secretion were analyzed as in Fig. 7; for both assays, +, normal function; –, reduced function.